Derivatives of gentamicin c1

ABSTRACT

DERIVATIVES OF GENTAMICIN C1 HAVE BEEN PREPARED WHICH POSSESS SUBSTANTIALLY IMPROVED ANTIBACTERIAL ACTIVITY. A EXAMPLE OF SUCH AN AGENT IS 1-(L-(-)-$-AMINO-AHYDROXYBUTYRYL)GENTAMICIN C1(IIIA,BB-K62).

United States Patent 3,780,018 DERIVATIVES 0F GENTAMICIN C Masataka Konishi, Yokohama, and Hiroshi Tsukiura, Tokyo, Japan, assignors to Bristol-Myers Company, New York, N.Y. No Drawing. Filed June 26, 1972, Ser. No. 266,170 Int. Cl. C07c 129/18 US. Cl. 260-210 AB 13 Claims ABSTRACT OF THE DISCLOSURE Derivatives of gentamicin C; have been prepared which possess substantially improved antibacterial activity. An example of such an agent is 1-[L-()-'y-amino-uhydroxybutyrylJgentamicin C [111a, BB K62].

BACKGROUND OF THE INVENTION (1) Field of the invention This invention relates to semisynthetic 1- or 2'-substituted derivatives of gentamicin C said compounds being prepared by acylating the 1 or 2-amino-function of gentamicin C with a -amino-u-hydroxybutyryl moiety.

(2) Description of the prior art (A) Gentamicin (gentamycin) and the fermentation thereof is the subject matter of US. Pat. Nos. 3,091,572 which issued May '28, 1963 and 3,136,704 which issued June 9, 1964. The patents describe the fermentation of gentamicin, complexes from Micromonospora purpurea, NN RL 2953 and Micromonospora echinospora, NNRL 2985. Variants thereof have also been deposited in the stock culture collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Ill. as M. echinospora var. ferruginea, NNRL 2995 and M. echinospora var. pallida, NNRL 2996.

(B) Gentamicin is further described in the Merck Index, 8th Edition, p. 485. The two major components, gentamicin C and C are described therein.

SUMMARY OF THE INVENTION The compound having the formula in which either R or R is L-(-)-y-amino-u-hydroxybutyryl and the other is hydrogen; or a nontoxic pharmaceutically acceptable acid addition salt thereof is a valuable antibacterial agent.

This nivention relates to semi-synthetic derivatives of gentamicin C, said compounds being known as 1 or 2'- 3,780,018 Patented Dec. 18, 1973 [L- -'y-amino-a-hydroxybutyryl] gentamicin C having the formula and in which either R or R is L-(-)-7-amino-a-hydroxybutyryl and the other is hydrogen, or a nontoxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceutically acceptable acid addition salt shall mean a mono, di-, tri-, tetra, or penta salt formed by the interaction of 1 molecule of compound IV with 1-5 moles of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine containing pharmaceuticals.

Gentamicin C is fermented as a complex of three closely related components designated 0,, C and C For the purpose of this application we are only concerned with gentamicin C the starting material of the compounds of the instant invention, which has the formula The compounds of the present invention are prepared by the following diagramatic scheme:

(A) Gentamicin C1 N-hydroxysuccinimide ester of L- -'y-benzyloxyearbonylamino-a-hydroxybutyric acid (11a) cx ua-i -o-ca g and/ or ea n o-Fa r es; Pin-Emu a ta s CH N J r 3. Compounds II lPd/G cH-NH-CH 2 5 cw; I'm

30-?3 on (Illa) a F *2 and $ 5 CK-NH-CH H N-OH -ca CH-C A preferred embodiment of he present invention is the compound having the formula CH3 CH-NH-Clij in which either R or R is L-()-'y-amino-a-hydroxybutyryl or L-()-'y-benzyloxycarbonylamino-a-hydroxybutyryl and the other is hydrogen; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

Another preferred embodiment is the compound of Formula IV wherein either R or R is L-()-'y-benzyloxycarbonylamino-a-hydroxybutyryl and the other is hydrogen.

A further preferred embodiment is the compound of Formula IV wherein either R or R is L-(-)-'y-amino-uhydroxybutyryl and the other is hydrogen.

A most preferred embodiment is the compound of Formula IV wherein R is L-()-' -amino-a-hydroxybutyryl and R is hydrogen; or the mono or disulfate salt thereof.

Another most preferred embodiment is the compound of Formula IV wherein R is L-()-'y-amino-a-hydroxybutyryl and R is hydrogen; or the mono or disulfate salt thereof.

The objectives of the present invention have been achieved, by the provision according to the present invention of the process for the preparation of the compound having the formula CH .CH-NH-CH;

in which either R or R is L-(-)-'yamino-ot-hydroxybutyryl and the other is hydrogen; or a nontoxic pharmaceutically acceptable acid addition salt thereof; which process comprises the consecutive steps of (A) Acylating gentamicin C with an acylating agent having the formula W-NH-CH CHz-JJH-PJ-M VII in which W is a radical selected from the group comprising but preferably M is a radical selected from the group comprising but preferably ON or the like but preferably 1:1 water-ethylene glycol dimethyl ether, to produce a compound of the formula CH -N 5 in which W is as above; and

(B) Removing the blocking group W from the compounds IIa and IIb by methods commonly known in the art, and preferably when W is a radical of the formula by hydrogenating the compounds II with hydrogen in the presence of a metal catalyst, preferabl selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, but preferably palladium, and most preferably palladium on charcoal, in a waterwater miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether, propyleneglycol dimethyl ether, or the like, but preferably 1:1 Water-dioxane, and preferably in the presence of a catalytic amount of glacial acetic acid to produce the compounds of Formula IV.

It should be apparent to those knowledgeable in the art that other agents can be used in the process above to acylate the amine functions of the intermediate compounds of the instant invention. This disclosure is meant to include all such acylating agents that produce labile amine blocking groups, said labile blocking groups commonly employed in the synthesis of peptides. The labile blocking groups must be readily removable by methods commonly known in the art. Examples of said labile blocking groups and their removal can be found in the review of A. Kapoor, J. Pharm. Sciences 59, pp. 1-27 (1970). Functional equivalents as acylating agent for primary amine groups would include corresponding carboxylic chlorides, bromides, acid anhydrides, including mixed anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, of alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester of thioester (e.g., with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with the gentamicin C derivative after first reacting said free acid with N,N'-dimethylchloroforminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/6, 360 (1965)] Or of alkynylamine reagent [cf. R. Buijile and H. G. Viehe,

Angew, Chem., International Edition 3, 582 (1964)], or of a ketenimine reagent [cf. C. L. Stevens and M. E. Munk, J. Amer. Chem. Soc. 80, 4065 (1958)] or of an isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)]. Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'-carbonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolides. The byproduct, imidazole, precipitates and may be separated and the imidazolide isolated, but this is not essential. These reactions are well-known in the art (cf. US. Pat. Nos. 3,079,314, 3,117,126 and 3,129,224 and British Pat. Nos. 932,644, 957,570 and 959,054).

1-[L-(--)-'y-amino a hydroxybutyryngentamicin C (IIIa, BBK62) and 2'-[L-() y-amino-ot-hydroxybutyryl]gentamicin C (IIIb, BBK) both possess excellent antibacterial activity that appears superior in some respects to gentamicin C against certain organisms.

Illustrated below are two tables showing the minimal inhibitory concentrations (MICs) of gentamicin C and Compounds IIIa and HR) against a variety of gram-positive and gram-negative bacteria as obtained by the Steers agar-dilution method (Table I) using Nutrient Agar Medium.

TABLE 1 In Vitro Antimicrobial Activity of Gentamieln C Derivatives Bristol Strain No. BB-KGO BB-K62 Gent. 0;

E. colt, NIHJ 1. 6 0. 8 0. 2 E colt, .Iuhl--- A15119 1.6 1.6 0.4 1. 6 1. 6 0. 4 1. 6 1. 6 0.2 0Z 0.8 o. 4 0. 2 0. 4 0. 8 0.1 E coli, K 12 1.6 1.6 0.2 1. 6 1. 6 0. 2 0.8 0.8 0. 1

67 0. 4 0.8 0.1 E. colt, J R/W677-. 25 1. 6 12. 6 K. pneumoniae, D-l 0. 4 0. 4 0. 2 K. pneumoniae, Type 22, No.

3038 A20680 25 1.6 25 S. marcescens A20019 1. 6 1. 6 0. 4 P. aeruginosa, D-15. 100 3. 1 P. aeruginosa, H9, D

P. aeruginosa 100 100 12. 5 P. aeruginosa A9930 1. 6 3. 1 0. 2 P. aerugi'rtosanh A15150 100 100 12. 5 P. aeruginosa A15194 100 100 12. 5 P. aeruginosa, GM- 100 100 100 P. aeruginosa, GM-R 100 10000 100 P. aemginoaa, H6, D-114- P. vulgaris A9436 0. 8 1. 6 0. 2 P. vulgaris A9526 0.8 1.6 0. 2 P. mirabil2's A9554 3. 1 6. 3 0. 8 P mtrabili A9900 1. 6 3. 1 0. 4 P morgam A9553 1. 6 3. 1 0. 4 P. morgama- A20031 1. 6 8. 1 O. 8 S. aureus, Smith 0. 4 0. 8 0. 1 S. aureus, 2091 SM-B. 3 6. 3 3. 1 0. 8 S. aureus, KM-R l A20239 1. 6 1. 6 0. 4 B. subtilis A9506 Mycobaeterium 607 6. 3 3. 1 1. 6 Mycobacterimn 607, KM-R 100 100 100 Mycobacterium 607, KM-R,

SM-R B 100 100 100 Mgcobacterium phlei 1. 6 3. 1 1. 6 Mycobacterium Tame 3. 1 3. 1 0. 8

1 KM-R is kanamyein resistant. 2 GM-R is gentamicin resistant. I Sm-B is streptomycin resistant.

Compound IIIa, BB-K62, the major acylation product, showed much higher activity than gentamicin C against the two gentamicin-resistant organisms (E. coli A20'683 and K. pneumoniae A20680) which are known to inactivate gentamicin C by 2"-adenylation. However, BB-K62 was about M144; as active as gentamicin C against the gentamicin-sensitive organisms, and the gentamicin-resistant strains of Pseudomonus were not inhibited by BB-K62.

Compound IIIb, BB-K60, which was the other major product in the reaction, showed similar antibacterial ac tivities to those of BB-K62 except for the lack of activity against the gentamicin-resistant strains of E. coli and K. pneumoniae.

Both of the major acylation derivatives showed relatively decreased activity against Pseudomonas species as compared with gentamicin C They were also inactive against the gentamicin-resistant strains of Pseudomonas.

The Compounds 111a and IIIb are valuable as antibacterial agents, nutritional supplements in animal feeds, therapeutic agents in poultry and animals, including man, and are especially valuable in the treatment of infectious diseases caused by gram-positive and gram-negative bacteria.

The Compounds 111a and HR are useful as an adjunctive treatment for preoperative sterilization of the bowel when administered orally. Both aerobic and anaerobic flora which are susceptible to these drugs are reduced in the large intestine. When accompanied by adequate mechanical cleansing, they are useful in preparing for colonic surgery.

The Compounds Hla and -IIIb are effective in the treatment of systemic bacterial infections when administered parenterally in the dosage range of about 250 mg. to about 3000 mg. per day in divided doses three or four times a day. Generally the compounds are etfective when administered at a dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

Gentamicin C is a complex of at least three major components diifering only in the R substituent found on the 5 position of the dideoxy-sugar moiety of the gentamicin nucleus as shown below:

C1- CHr-NH1 Gentamicin C was obtained from the complex by the following procedure.

Separation of gentamicin C complex Gentamicin C complex (4.10 g.) as the free base was fractionated using a preparative counter current distribution (CCD) apparatus (100 ml. x 50 tubes) with a solvent system of CHCl -MeOH 17% aqueous NH OH (2: 1:1) to give the fractions shown below.

Amount Identifica- Fraction Tube No. recovered, g. tion (TLC 25-33 1. 46 0 main. 32-42 0. 72 01 C: 43-47 1. 20 C2+C1a 48-50 0. 87 Cla main.

1 Cellulose thin-layer plate, solvent system: lower layer of CHCls- %\ge2g)H17% NH4OH (2:1:1), R1: gentamicin G (0.69), C: (0.39), 01.

Fractions I and 11 were combined and distributed again by the same CCD system as above to give 194 g. of the gentamicin C component which was further purified by a Dowex 1 x 2 column chromatography yielding 1.70 g. of substantially pure gentamicin C Other fractions were purified similarly by CCD and Dowex 1 x 2 chromatography to give 1.13 g. of gentamicin C and 0.62 g. of gentamicin C EXAMPLES Example 1.Preparation of L-(-)-v-benzyloxycarbonylamino-m-hydroxybutyric acid (VI) L-()-'y-amino-u-hydroxybutyric acid (7.4 g., 0.062 mole) was added to a solution of 5.2 g. (0.13 mole) of sodium hydroxide in 50 ml. of water. To the stirred solution was added dropwise at 0-5 C. over a period of 0.5 hour, 11.7 g. (0.068 mole) of carbobenzoxy chloride and the mixture was continued to stir for one hour at the same temperature. The reaction mixture was washed with 50 ml. of ether, adjusted to pH 2 with dilute hydrochloric acid and extracted with four -ml. portions of ether. The ethereal extracts were combined, washed with a small amount of saturated sodium chloride solution, dried with anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the resulting residue was crystallized from benzene to give 11.6 g.

(74%) of colorless plates; melting point 78.5-79.5 C., [0:1 5:45 (c. =2, CH OH). Infrared (IR) [KBr]: IR(KBr) 'y 1740, 1690 cmr Nuclear Magnetic Resonance (NMR) (acetone-d 5(in p.p.m. from TMS) 2.0 (2H, m.), 3.29 (2H, d-d, J=6.7 and 12 Hz.), 4.16 (1H, d-d, 1:45 and 8 Hz.), 4.99 (2H, s.), 6.2 (2H, broad), 7.21 (5H, s.).

Analysis.-Calcd for C H NO (percent): C, 56.91; H, 5.97; N, 5.53. Found (percent): C, 56.66; H, 5.97; N, 5.47.

Example 2.-N-hydroxysuccinimide ester of L-(--)-'ybenzyloxycarbonylamino-a-hydroxybutyric acid (VII) A solution of 10.6 g. (0.042 mole) of VI and 4.8 g. (0.042 mole) of N-hydroxysuccinimide in 200 ml. of ethyl acetate was cooled to 0 C. and then 8.6 g. (0.042 mole) of dicyclohexylcarbodiimide was added. The mixture was kept overnight in a refrigerator. The dicyclohexylurea which separated was filtered off and the filtrate was concentrated to about 50 ml. under reduced pressure to give colorless crystals of VII which were collected by filtration; 6.4 g., M.P. 121-1225 C. The filtrate was evaporated to dryness in vacuo and the crystalline residue was washed with 20 ml. of a benzene-n-hexane mixture to give an additional amount of VII. The total yield was 13.4 g. (92%). [111 1.5 (c.-=2, CHCI IR (KBr) 7 1810, 1755, 1740, 1680 cmr NMR (acetone-d 6 (in p.p.m. from TMS) 2.0, (2H, m.), 2.83 (2H, s.), 3.37 (2H, d-d, I=6.5 and 12.5 Hz.), 4.56 (1H, m.), 4.99 (2H, s.), 6.3 (2H, broad), 7.23 (5H, s.).

Analysis.Calcd for C H N O (percent): C, 54.85; H, 5.18; N, 8.00. Found (percent): C, 54.79; 54.70; H, 5.21, 5.20; N, 8.14, 8.12.

Example 3.Preparation of 1-[L-()-'y-benzyloxycarbonylamino a hydroxybutyryl] gentamicin C (11a) and 2' [L-()-'y-benzyloxycarbonylamino a hydroxybutyryl] gentamicin C (11b) To a stirred solution of 1.10 g. (2.2 mmoles) of gentamicin C in 50 ml. of 50% TH-F (tetrahydrofuran) and water solution was added 770 mg. (2.2 mmoles) of N hydroxysuccinimide ester of L-(-) 'y benzyloxycarbonylamino a hydroxybutyric acid at 5 C. The mixture was continued to stir for 20 hours at the same temperature and concentrated in vacuo to dryness. The product thus obtained was separated into two fractions by distribution between 30 ml. of n-butanol and 30 ml.

1 G. W. Anderson et 8.1., J. Am. Chem. 800., 86, 1839 (1964).

9 of water. The n-butanol layer which contained acylated gentamicin C was concentrated in vacuo to give 697 mg. of white solid determined to be primarily a mixture of compounds IIa and 11b. A mixture of intact gentamicin C and N-hydroxysuccimide (1.052 g.) was recovered from the aqueous layer.

Example 4.Preparation of l-[L-() 'y amino-whydroxybutyryl]gentamicin C (IIIa) and 2'-[L-()-'yamino-a-hydroxybutyryl] gentamicin C (Illb) The acylated crude mixture of IIa and Rh obtained in Example 3 above (680 mg.) was dissolved in 30 ml. of 50% ethanol and hydrogenated under atmospheric pressure in the presence of 500 mg. of palladium on charcoal overnight at room temperature. The mixture was filtered and the clear filtrate was concentrated in vacuo. The concentrate was loaded on a resin column of Amberlite CG-SO (NH 60 ml.). The column was 10 30.1 g. (56%) of a diastereomer of the dehydroabiethylamine salt. M.P. 93-94 C. +15 (c. 2.5, MeOH). Recrystallization from 300 ml. of ethanol gave 23.2 g. (43%) of the pure product. M.P. 94-95 C. +10.8 (c. 2.5, MeOH). Further recrystallization did not change the melting point and the specific rotation.

Analysis.Calcd for C H N O H O (percent): C, 69.54; H, 8.02; N, 5.07. Found (percent): C, 69.58; H, 8.08; N, 5.07.

(B) L-(-)w-amino-a-hydroxybutyric acid: To a golution of 1.5 g. (0.014 mole) of sodium carbonate in ml. of water were added 5.3 g. (0.01 mole) of dehydroabietylammonium L-m-hydroxy 'y phthalimidobutyrate and 60 ml. of ether. The mixture was shaken vigorously until all of the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with 20-ml. portions of ether and evaporated to 15 ml. under reduced pressure. To the concentrate was added 10 ml. of concentrated hydrochloric acid and the mixture was refluxed for ten hours. After cooling, separated phthalic acid was removed by filtration. The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml. of water and the solution was evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The residual syrup was dissolved in 10 ml. of water and filtered to remove a small 1 TLC=Silica gel plate. CHCl -MeOH-28% NH4OH-Hz0 (124:2:1),

Analysis.-Calcd for C H O N 3/2H CO (percent): C, 47.38; H, 7.95; N, 12.51. Found for BB-K60 (percent): C, 47.14; H, 7.39; N, 11.86. Found for BB- K62 (percent): C, 47.02; H, 7.38; N, 12.18.

The identity of BB-K61 was not established although it is known to be a monoacylated-gentamicin C derivative with the L-(- )-'y-amino-a-hydroxy=butyryl moiety on one of the amine functions other than the 1 or 2 positions. The compound has low antibacterial activity as compared to compounds Illa or IIIb.

Example 5 .-Preparation of L-(-)-'y-amino-u-hydroxybutyric acid from ambutyrosin A or B or mixtures thereof Example 6.-Prep-aration of L-()-'y-amino-u-hydroxybutyric acid from DL-ot-hydroxy-y-phthalimidobutyric acid (A) Dehydroabiethylammonium L u hydroxy-vphthalimidobutyrate: To a solution of 25 g. (0.1 mole) of 2-hydroxy-v-phthalimidobutyric acid in 200 ml. of ethanol was added a solution of 29 g. (0.1 mole) of dehydroabietylamine in 130 ml. of ethanol. The solution was shaken vigorously for a minute and stood at room temperature for five hours during which time fine needles crystallized out. The crystals were collected by filtration, 'washed with 50 m1. of ethanol and air dried to obtain 2 Y. Suito et 211., Tetrahedron Letters, 1970, 4863.

amount of insoluble phthalic acid. The filtrate was adsorbed on a column of IR-120' (H 1 cm. x 35 cm.), the column was Washed with 300 ml. of water and eluted with 1 N ammonium hydroxide solution. The eluate was collected in 15-ml. fraction. The ninhydrin positive fractions 10 to 16 were combined and evaporated under reduced pressure to give a syrup which crystallized gradually. The crystals were triturated with ethanol, filtered and dried in a vacuum desiccator to give 0.78 g. (66%) of L-(-)-'y-amino-a-hydroxybutyric acid M.P. 206-207 C. -29 (c. 2.5, H 0). The IR spectrum was identical with the authentic sample which was obtained from ambutyrosin.

Example 7.Preparation of the monosplfate salt of 1- [L-( )-'y-amino-a-hydroxybutyryl]gentamicin C or 2'-[L- -'y-amino-a-hydroxybutyryl] gentamicin C One mole of 1-[L-(--)-'y-amino-u-hydroxybutyryl] gentamicin C (lIIa) or 2-[L-()-'y-amino-a-hydroxybutyryl] gentamicin C (Illb) is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added one mole of sulfuric acid dissolved in 500 ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture till precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate.

Example 8.--Preparation of the disulfate salt of 1 or 2- [L- -'y-amino-u-hydroxybutyryl] gentamicin C One mole of either 1-[L-( -amino-a-hydroxybutyryl]gentamicin C or 2 '-[L-( )-'y-amino-ot-hydroxybutyrylJgentamicin C is dissolved in 1 to 3 liters of water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added 2 moles of sulfuric acid dissolved in ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture until precipitation occurs. The solids are collected by filtration and are determined to be the desired disulfate salt.

1 1 We claim: 1. A compound having the formula CH3 cit-meets;

in which either R or R is L-()- -amino-a-hydroxybutyryl or L-(-)-'ybenzyloxycarbonylamino-a-hydroxybutyryl and the other is hydrogen; or a nontoxic pharma ceutically acceptable acid addition salt thereof.

2. The compound of claim 1 wherein either R or R is L-()-'y-benzyloxycarbonylamino-a hydroxybutyryl and the other is hydrogen.

3. The compound of claim 1 wherein either R or R is L-(-)-'y-arnino a hydroxybutyryl and the other is hydrogen.

4. The compound of claim 1 wherein R is H and R is L-(-)-'y-amino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

5. The compound of claim 1 wherein R is H and R is L-()-'y-amino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

6. The monosulfate salt of the compound of claim 4.

7. The monosulfate salt of the compound of claim 5.

8. The disulfate salt of the compound of claim 4.

9. The disulfate salt of the compound of claim 5.

10. The mono or polyhydrates of the compound of claim 3.

11. The mono or polyhydrates of the compound of claim 4.

12. The mono or polyhydrates of the compounds of claim 6.

13. The mono or polyhydrates of the compounds of claim 8.

References Cited UNITED STATES PATENTS JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R. 

